Transferring cargo into mammalian cells over a wide range of 1 sizes, including proteins, DNA, RNA, chromosomes, nuclei, and inanimate particles, such as quantum dots, surface-enhanced Raman scattering (SERS) particles, and microbeads, is highly desirable in many fields of biology. Delivery methods, such as endocytosis, can entrap cargo in an endosome, where the low pH microenvironment and lytic enzymes often lead to cargo degradation (Luo and Saltzman (2000) Nat. Biotechnol. 18: 33-37). Viral and chemical delivery methods package the cargo inside a virus or form chemical complexes that enhance uptake (Naldini et al. (1996) Science, 272: 263-267; Feigner et al. (1987) Proc. Natl. Acad. Sci. USA, 84: 7413-7417). However, toxicity, cell-type specific uptake, and more importantly limited cargo packing capacity impose a significant constraint on cargo size and transferable cell types (Luo and Saltzman, supra.).
Physical transfer methods include electroporation (Chu, et al. (1987) Nucleic Acids Res. 15: 1311-1326) and sonoporation (Mitragotri (2005) Nat. Rev. Drug Discovery, 4: 255-260), which produce randomly distributed nanoscale pores, and optoporation (Tirlapur and Konig (2002) Nature, 418: 290-291; Vogel, et al. (2005) Appl. Phys. B: Laser Opt., 81: 1015-1047; Clark et al. (2006) J. Biomed. Opt., 11: 014034), which generates pores on the cell membrane at the laser focal point. Through these pores, small cargo is delivered into cells by thermal diffusion or by an electric field. Delivery of large cargo with these methods has low efficiency due to the slow speed of cargo diffusion and decreasing cell viability with increasing pore size (Stevenson et al. (2006) Opt. Express, 14: 7125-7133). Microcapillary injection (King (2004) Methods in Molecular Biology 245: Gene Delivery to Mammalian Cells 1; Humana Press Inc.: Totowa, N.J.) uses a sharp lass tip to mechanically penetrate a cell membrane for delivery. However, mechanical trauma from membrane penetration limits the typical pipet tip to 0.5 um in diameter in order to maintain cell viability (Han et al. (2998) J. Nanomed. Nanotechnol. Biol. Med., 4: 215-225).
Cargo larger than the pipet tip cannot be injected due to pipet clogging and cargo shearing. Electro-injection, which combines electroporation with microcapillary injection, has demonstrated small molecule delivery, such as RNA and plasmid DNA, into live cells (Boudes et al. (208) J. Neurosci. Meth., 170: 204-211; Kitamura et al. (2008) Nat. Meth., 5: 61-67) and bacteria delivery into artificial lipid vesicles (Hurtig and Orwar (2008) Soft Matter, 4: 1515-1520) by weakening the contacting cell membrane with an electric field, followed by gentle mechanical penetration into the cell. Alternatively, a simple lipid assisted microinjection (SLAM) technique (Laffafian and Hallett (1998) Biophys. J., 75: 2558-2563) incorporates synthetic lipid molecules at the tip of a glass microcapillary. Contact of the SLAM micropipette with a cell membrane allowed the lipid molecules to fuse with the cell membrane to form a continuous and temporary pathway for cargo delivery. This method avoids the zigzag stabbing motion of the micropipette tip through the cell membrane. However, the lipophilic interactions with cargo and cell membrane can produce unwanted biological effects in the cell as well as with the delivery cargo, limiting this method to specific cell types and cargo contents.